A case of hepatitis B reactivation due to the hepatitis B virus escape mutant in a patient undergoing chemotherapy

A 62-year-old man had chronic hepatitis B virus (HBV) infection and was diagnosed with liver cirrhosis. At the time of diagnosis the patient’s virologic markers were positive for hepatitis B surface antigen (HBsAg), antibody to hepatitis B e antigen (anti-HBe) and antibody to hepatitis B core antigen (anti-HBc), while antibody to hepatitis B surface antigen (anti-HBs) and HBV DNA were negative. Later the patient received chemotherapy for malignancy. However, this was interrupted due to elevated liver enzymes. At the same time HBV DNA became positive. Lamivudine (LMV) therapy was administered immediately. However, the levels of serum aminotransferase and total bilirubin (TB) were still rising. Finally the patient died of fulminant hepatic failure. A sequence revealed HBV genotype C (HBsAg subtype adw) with immune escape mutations, F8L, S34L, F41S, G44V, F93C, V96G, L110I, C149Y and F161Y. The high morbidity and mortality of this complication is one of the major obstacles to completing the standard treatment for malignancy in HBV carriers. Therefore, the relative risk of antiviral prophylactic failure should be further assessed and the optimal strategy for antiviral prophylaxis in HBsAg-positive patients with oncologic and hematologic malignancies undergoing chemotherapy should be revised.     

Endocytosis of hepatitis B immune globulin into hepatocytes inhibits the secretion of hepatitis B virus surface antigen and virions

Hepatitis B immunoglobulin is used for prophylaxis against hepatitis B virus (HBV) and is thought to act by neutralization of virions and hepatitis B virus surface antigen (HBsAg)-containing particles in circulation. Using a panel of hepatocyte-derived cell lines, the present study investigated in vitro whether HBs-specific immunoglobulin G (IgG) is internalized in hepatocytes and whether it interacts with HBsAg in the cells. By immunoelectron microscopy and immunoblotting, human IgG and FcRn receptor for IgG were demonstrated on cellular membranes and in cytoplasmic extracts, irrespective of the HBsAg status of the cells. Furthermore, HBsAg and anti-HBs were shown to be colocalized in the same cellular compartment by two-color confocal microscopy. Endocytosis of HBs-specific IgG caused intracellular accumulation of HBsAg in a dose-dependent manner and inhibited the secretion of HBsAg and HBV virions from the cells. These effects were not observed with F(ab)(2) fragments or nonimmune IgG as controls. The specificity of intracellular HBsAg- anti-HBs interaction was further investigated in cells transfected with HBV genomes expressing wild-type HBsAg or immune escape HBsAg (with a G145R mutation). Monoclonal anti-HBs markedly reduced the secretion of wild-type HBsAg, while the secretion of mutant HBsAg was not affected. These results suggest that HBs-specific IgG binds to hepatocytes and interacts with HBsAg within the cells. This may be relevant for the selection of surface antibody escape mutations.     

Amino acid substitutions at positions 122 and 145 of hepatitis B virus surface antigen (HBsAg) determine the antigenicity and immunogenicity of HBsAg and influence in vivo HBsAg clearance

A variety of amino acid substitutions, such as K122I and G145R, have been identified around or within the a determinant of hepatitis B surface antigen (HBsAg), impair HBsAg secretion and antibody binding, and may be responsible for immune escape in patients. In this study, we examined how different substitutions at amino acid positions 122 and 145 of HBsAg influence HBsAg expression, secretion, and recognition by anti-HBs antibodies. The results showed that the hydrophobicity, the presence of the phenyl group, and the charges in the side chain of the amino acid residues at position 145 reduced HBsAg secretion and impaired reactivity with anti-HBs antibodies. Only the substitution K122I at position 122 affected HBsAg secretion and recognition by anti-HBs antibodies. Genetic immunization in mice demonstrated that the priming of anti-HBs antibody response was strongly impaired by the substitutions K122I, G145R, and others, like G145I, G145W, and G145E. Mice preimmunized with wild-type HBsAg (wtHBsAg) or variant HBsAg (vtHBsAg) were challenged by hydrodynamic injection (HI) with a replication-competent hepatitis B virus (HBV) clone. HBsAg persisted in peripheral blood for at least 3 days after HI in mice preimmunized with vtHBsAg but was undetectable in mice preimmunized with wtHBsAg, indicating that vtHBsAgs fail to induce proper immune responses for efficient HBsAg clearance. In conclusion, the biochemical properties of amino acid residues at positions 122 and 145 of HBsAg have a major effect on antigenicity and immunogenicity. In addition, the presence of proper anti-HBs antibodies is indispensable for the neutralization and clearance of HBsAg during HBV infection.     

Paradoxical HBsAg and anti-HBs coexistence among Chronic HBV Infections: Causes and Consequences

Hepatitis B surface antigen (HBsAg) and Hepatitis B surface antibody (anti-HBs) were reported simultaneously among Hepatitis B virus (HBV) infections. HBsAg is a specific indicator of acute or chronic HBV infections, while anti-HBs is a protective antibody reflecting the recovery and immunity of hosts. HBsAg and anti-HBs coexist during seroconversion and then form immune complex, which is rare detected in clinical cases. However, with the promotion of vaccination and the application of various antiviral drugs, along with the rapid development of medical technology, the coexistence of HBsAg and anti-HBs has become more prevalent. Mutations in the viral genomes, immune status and genetic factors of hosts may contribute to the coexistence. Novel HBsAg assays, with higher sensitivity and ability to detect mutations or immune complexes, can also yield HBsAg/anti-HBs coexistence. The discovery of coexistence has shattered the idea of traditional serological patterns and raised questions about the effectiveness of vaccines. Worth noting is that HBsAg/anti-HBs double positivity is strongly associated with progressive liver diseases, especially hepatocellular carcinoma. In conclusion, viral mutations, host factors, and methodology impacts can all lead to the coexistence of HBsAg and anti-HBs. This coexistence is not an indicator of improvement, as an increased risk of adverse clinical outcomes still exists.     

A case of hepatitis B reactivation in an anti-HBs positive, anti-HBc positive non-Hodgkin’s lymphoma patient

Dear Editor, We report a case of HBV reactivation in an anti-HBs positive,anti-HBc positive non-Hodgkin's lymphoma patient.Hepatitis B virus (HBV) reactivation is a well-recognized complication of patients undergoing chemotherapy or immunosuppressive therapy for lymphomas.The presence of antibodies to the hepatitis B surface antigen (anti-HBs) has been identified to be a factor preventing HBV reactivation in patients with occult HBV infection receiving chemotherapy.In this paper,we present a non-Hodgkin Lymphoma patient who,before immunosuppressive therapy,displayed positive anti-HBs and positive antibodies to hepatitis B core antigens (anti-HBc),as markers of resolved HBV infection,and developed hepatitis B surface antigen (HBsAg) and high viraemia with an HBV escape mutant after rituximabbased administration.The sequencing data revealed HBV genotype D with two known escape mutations,P120S.     

甘肃省1～59岁人群乙型肝炎病毒感染的血清流行病学调查

为了解甘肃省现阶段乙型肝炎病毒感染的现状,分析乙肝病毒感染血清学指标的变化,采用多阶段整群抽样的方法,抽取5个县区1~59岁人群共2200人进行调查。以ELISA方法对血清标本统一检测乙肝病毒表面抗原(HBsAg)、乙肝病毒表面抗体(抗-HBs)、乙肝病毒核心抗体(抗-HBc)。结果显示,甘肃省1~59岁调查人群HBsAg阳性率为3.59%,抗-HBs和抗-HBc阳性率分别为49.45%、16.33%;HBV总感染率为27.50%。比1992年HBV总感染率下降了36.48%,HBsAg阳性率下降了45.94%;1~4岁儿童HBsAg阳性率仅为1.13%,比1992年(5.34%)下降78.84%。甘肃省1~59岁人群乙肝病毒感染率下降,抗-HBs阳性率升高,尤其在1~4岁儿童变化更为明显;甘肃省乙肝感染逐步呈现由中高流行转向低流行区的趋势;乙肝疫苗免疫取得显著效果。     

Detection of hepatitis B virus DNA by polymerase chain reaction in HBsAG negative Senegalese patients suffering from cirrhosis or primary liver cancer

The polymerase chain reaction was used to search for hepatitis B virus (HBV)-DNA sequences in the sera of HBsAg-negative Senegalese patients suffering from liver cirrhosis or liver cancer. Amplified HBV-DNA sequences were detected by hybridization with a digoxigenin-labelled HBV-DNA probe. HBV-DNA was detected in 17% of HBsAg negative Senegalese subjects from the general population and in 44% and 58% of the patients suffering from cirrhosis or primary hepatocellular carcinoma (PHCC) respectively. In the control group, amplified HBV-DNA was detected in 25% of the subjects without HBsAg and anti-HBs antibodies, and in 6% of subjects positive for anti-HBs antibodies. This study confirmed the hypothesis that there is an etiologic link between HBV and PHCC in HBsAg-negative patients.     

Clinical and Virological Characteristics of Chronic Hepatitis B Patients with Coexistence of HBsAg and Anti-HBs

Coexistence of hepatitis B surface antigen (HBsAg) and antibody against HBsAg (anti-HBs) comprises an atypical serological profile in patients with chronic hepatitis B virus (HBV) infection. In this study, in total 94 patients with coexisting HBsAg and anti-HBs and 94 age- and sex-matched patients with positive HBsAg were characterized by quantitatively measuring HBsAg and HBV DNA, sequencing large S genes, and observing clinical features. Compared with common hepatitis B patients, the patients with coexisting HBsAg and anti-HBs had lower HBsAg and HBV DNA levels. These two groups had similar rate of pre-S deletion mutations. However, in patients with coexisting HBsAg and anti-HBs, more amino acid substitutions in the a determinant of S gene were observed in HBV genotype C, but not in genotype B. Fourteen patients with coexisting HBsAg and anti-HBs were followed up for an average of 15.5 months. There were no significant changes in the levels of HBsAg, anti-HBs, HBV DNA and ALT over the follow-up period. Compared with the baseline sequences, amino acid substitutions in the MHR of HBsAg occurred in 14.3% (2/14) patients. In conclusion, coexistence of HBsAg and anti-HBs may be associated with higher frequency of mutations in the a determinant of HBV genotype C.     

Neutralization of hepatitis B virus surface antigen with immunoglobulin preparations

The in vitro experiments with immunoglobulin and blood plasma containing hepatitis B virus (HBV) markers, revealed that the control of immunoglobulin preparations for the presence of hepatitis B virus surface antigen (HBsAg), mandatory for Russia, was not sufficiently informative. The neutralization of HBsAg with specific antibodies to the level, not determined by the EIA method, reached not less than 24 ng/ ml in 2 hours of incubation and not less than 49 ng/ml in 24 hours of incubation, which, when evaluated in 1 lU of anti-HBs, was 34.6 +/- 0.9 ng and 70.7 +/- 1.8 ng of HBsAg respectively. The process of the formation of immune complexes depended mainly on the time of incubation of experimental samples and on the antibody--antigen proportion in the system. The neutralization of viruses by antibodies had no influence on the capacity of the polymerase chain reaction to detect HBV DNA.     

免疫预防阻断乙型肝炎病毒母婴传播效果的观察

阻断乙型肝炎病毒(HBV)母婴传播是控制乙型肝炎的重大问题。为探讨免疫预防对阻断HBV母婴传播的效果及影响因素,对667例HBV表面抗原(HBsAg)阳性孕妇及其婴儿进行研究。这些孕妇按HBVe抗原(HBeAg)和HBVDNA检测结果,分为HBeAg阳性组及阴性组、DNA阳性组及阴性组;按是否于孕晚期注射乙型肝炎免疫球蛋白(HBIG),分为注射组及未注射组。婴儿于出生24h内均肌内注射HBIG100IU,并按0、1、6方案注射10μg重组酵母HBV疫苗;8～12月龄后随访婴儿,并进行HBV标志物(HBV-M)检测。667个婴儿中,20例感染HBV,免疫阻断失败率为3.0%。孕妇HBeAg阳性组免疫阻断失败率为8.7%,阴性组为0.2%,两组差异显著(P<0.001);两组婴儿对疫苗免疫应答率分别为83.0%和83.1%,无显著差异(P=0.988)。孕妇DNA阳性组免疫阻断失败率为8.1%,HBVDNA均≥6log10copies/ml。孕期注射与未注射HBIG组婴儿免疫阻断失败率分别为3.7%和2.7%,无显著差异(P=0.479);两组婴儿对疫苗免疫应答率分别为84.4%和82.4%,无显著差异(P=0.519)。孕妇HBeAg阳性注射HBIG组与未注射组的免疫阻断失败率分别为8.4%和8.9%,无显著差异(P=0.892)。孕妇HBeAg阴性注射与未注射HBIG组的免疫阻断失败率分别为0.0%和0.3%,也无显著差异(P=0.538)。11例免疫阻断失败的婴儿中,10例出生时血清HBsAg已为阳性;8～12个月后随访,HBsAg仍持续阳性,提示为宫内感染。本研究证实,孕期注射HBIG未能提高婴儿对HBV疫苗加HBIG的免疫阻断效果。宫内感染可能是疫苗加HBIG免疫阻断失败的主要原因。采用降低孕妇血清HBVDNA的措施,如对孕妇进行抗HBV治疗,也许能降低HBV宫内感染率。     

Antigenic structure of human hepatitis A virus defined by analysis of escape mutants selected against murine monoclonal antibodies.

We examined the antigenic structure of human hepatitis A virus (HAV) by characterizing a series of 21 murine monoclonal-antibody-resistant neutralization escape mutants derived from the HM175 virus strain. The escape phenotype of each mutant was associated with reduced antibody binding in radioimmunofocus assays. Neutralization escape mutations were identified at the Asp-70 and Gln-74 residues of the capsid protein VP3, as well as at Ser-102, Val-171, Ala-176, and Lys-221 of VP1. With the exception of the Lys-221 mutants, substantial cross-resistance was evident among escape mutants tested against a panel of 22 neutralizing monoclonal antibodies, suggesting that the involved residues contribute to epitopes composing a single antigenic site. As mutations at one or more of these residues conferred resistance to 20 of 22 murine antibodies, this site appears to be immunodominant in the mouse. However, multiple mutants selected independently against any one monoclonal antibody had mutations at only one or, at the most, two amino acid residues within the capsid proteins, confirming that there are multiple epitopes within this antigenic site and suggesting that single-amino-acid residues contributing to these epitopes may play key roles in the binding of individual antibodies. A second, potentially independent antigenic site was identified by three escape mutants with different substitutions at Lys-221 of VP1. These mutants were resistant only to antibody H7C27, while H7C27 effectively neutralized all other escape mutants. These data support the existence of an immunodominant neutralization site in the antigenic structure of hepatitis A virus which involves residues of VP3 and VP1 and a second, potentially independent site involving residue 221 of VP1.     

钦南区1～6岁儿童乙肝及白喉疫苗免疫效果分析

目的分析钦南区儿童乙肝及白喉疫苗接种后的免疫效果,为该地区儿童免疫规划工作提供科学依据。方法采用整群随机抽样方法,选取5个乡镇1~6岁常住儿童151名为调查对象,进行病毒性乙型肝炎(乙肝)、白喉血清学检测,并对结果进行分析。结果 HBsAg阳性2人,阳性率1.32%;抗-HBs阳性112人,阳性率74.17%。白喉IgG阳性142人,阳性率94.04%。抗-HBs中位数21.32 mIU/mL,白喉IgG中位数0.14 mIU/mL,乙肝疫苗首针及时接种率83.44%;男女抗-HBs阳性率、白喉IgG阳性率、抗体中位数差异均无统计学意义(P0.05),抗-HBs阳性率、白喉IgG阳性率随年龄的增长而下降(P0.01)。结论钦南区1~6岁儿童乙肝疫苗接种率、首针及时接种率均达到了国家免疫规划的目标,白喉IgG阳性率维持较高的水平,但HBsAg阳性率略高于国家免疫规划的目标,儿童免疫规划工作仍需进一步加强。     

HBV-DNA与乙型肝炎血清标志物的相关性分析

目的：探讨乙型肝炎病毒（HBV）DNA载量与其血清标志物的相关性。方法：运用荧光定量聚合酶链反应（FQ-PCR）、酶联免疫吸附实验（ELISA）分别检测503例患者HBV．DNA载量和HBV血清标志物。根据HBV血清标志物结果分为大三阳组、小三阳组、少见模式组、抗体阳性及全阴组,比较各组间HBV—DNA的阳性率及定量值。结果：在大三阳组、小三阳组、少见模式组、抗体阳性及全阴组HBV—DNA的阳性率分别为90％、65．1％、65．2％、2．0％,HBV—DNA的定量结果（10gHBV—DNA）别为6．32±1．96、2．01±1．68、3．48＋2．52f抗体阳性及全阴组阳性例数过低,不纳入统计）。大三阳组HBV—DNA的阳性率显著高于小三阳组（P〈0．05）。大三阳组、小三阳组HBV—DNA的阳性率与少见模式组比较,差异均无统计学意义（P〉0．05）,但大三阳组、小三阳组、少见模式组HBV．DNA的阳性率均显著高于抗体阳性及全阴组（P〈0．01）。HBsAg、HBeAg阳性组HBV—DNA的阳性率分别显著高于HB—sAg、HBeAg阴性组（P〈0．01）。小三阳组、少见模式组HBV．DNA载量均显著低于大三阳组（P〈0．01）,少见模式组HBV—DNA载量显著高于小三阳组（P〈0．05）。结论：HBV—DNA的阳性率与HBeAg、HBsAg相关;HBV—DNA栽量与HBV血清标志物模式相关。     

Impaired humoral immune response to hepatitis B vaccine in patients on maintenance hemodialysis

Hepatitis B virus (HBV) infection is a worldwide health problem. We aimed in this study to investigate the humoral immune response derived to HBV vaccine following completing the vaccine series in Madinah. Two hundred and two Saudi hemodialysis (HD) patients were included in this cross-sectional study. Mean concentration of Hepatitis B surface antibody (anti-HBs) was significantly higher among patients who received the vaccination twice compared to patients who received the vaccination only after starting hemodialysis (252 ± 489 mIU/mL vs. 144 ± 327 mIU/mL, respectively, p = 0.008). Almost half of the study sample were non-protected and showed anti-HBs concentration < 10 mlU/mL. In contrast, 20.3% (n = 41) were identified as poor responders (10–100 mlU/mL) and only 28.2% (n = 57) were identified as good responders (10–100 mlU/mL). However, the latter two groups were accounted as protected (48.5%, n = 98). Patients sex was associated with anti-HBs concentration (non-responders; poor responders; good responders), where significantly higher proportion of good responders were females compared to males (p = 0.007). In conclusion, HBV vaccine is efficient to elicit humoral immune response in hemodialysis patients.     

抗原抗体复合物型治疗性疫苗(乙克)临床研究中患者血清乙型肝炎病毒表面抗原下降的分析与启示

近年来全球慢性乙型肝炎(chronic hepatitis B,CHB)防治指南提出了“功能性治愈”(functional cure)的概念,即患者经过治疗达到血清乙型肝炎病毒表面抗原(hepatitis B virus surface antigen,HBsAg)消失,但现有抗病毒治疗很难实现这一目标。本研究对既往临床试验中经抗原抗体复合物型治疗性疫苗(乙克)治疗后的CHB患者HBsAg下降情况进行了归纳分析,结果显示,经乙克治疗随访后达到乙型肝炎e抗原(hepatitis B e antigen,HBeAg)血清学转换者的HBsAg下降高达0.95log₁₀IU/mL,显著高于未达到HBeAg血清学转换者的0.32log₁₀IU/mL(P<0.01),而经氢氧化铝佐剂治疗随访后发生HBeAg血清学转换(0.49log₁₀IU/mL)者与未发生HBeAg血清学转换者(0.36log₁₀IU/mL)之间HBsAg下降无统计学差异。乙克组治疗过程中,丙氨酸氨基转移酶(alanine aminotransferase,ALT)骤升(ALT flare)在HBsAg下降>1.0log₁₀IU/mL者中较多见,氢氧化铝组未观察到此现象。回归分析显示,乙克治疗后HBsAg下降的影响因素有患者出现HBeAg血清学转换、感染的HBV为B基因型、治疗过程中ALT出现10倍增高,以及基线血清HBsAg为高水平。结果提示,乙克诱导的特异性免疫对降低CHB患者血清HBsAg水平有一定效果,采用“抗病毒药物治疗＋针对HBsAg的中和性抗体被动免疫＋乙克主动免疫”的“三明治”治疗策略可能会提高“功能性治愈”率。     

Priming of immune responses to hepatitis B surface antigen in young mice immunized in the presence of maternally derived antibodies

Early vaccination is necessary to protect infants from various infectious diseases. However, this is often unsuccessful largely due to the immaturity of the neonatal immune system. Furthermore, maternally derived antibodies can interfere with active immunization. We have previously shown in young mice that immune responses against several different antigens can be improved by the addition of oligodeoxynucleotides containing immunostimulatory CpG motifs (CpG ODN). In this study we have evaluated immunization of newborn (1-7-day-old) BALB/c mice against hepatitis B surface antigen (HBsAg), with alum and/or CpG ODN, in the presence of high levels of maternal antibody against HBsAg (anti-HBs). Seroconversion rates and anti-HBs titers were compared to those induced by a HBsAg-expressing plasmid, since other studies had suggested DNA vaccines to be superior to protein vaccines in young mice with maternal antibody. HBsAg/alum/CpG ODN was superior to DNA vaccine in inducing HBsAg-specific CTL responses in young mice in the presence of maternally transferred anti-HBs antibodies. However, B cell responses to both HBsAg/alum/CpG ODN and DNA vaccines remained weak in the presence of maternally transferred anti-HBs antibodies.     

Naturally occurring escape mutants of hepatitis B virus with various mutations in the S gene in carriers seropositive for antibody to hepatitis B surface antigen.

Hepatitis B virus (HBV) DNA was extracted from sera of six carriers with hepatitis B e antigen as well as antibody to hepatitis B surface antigen and sequenced within the pre-S regions and the S gene. HBV DNA clones from five of these carriers had point mutations in the S gene, resulting in conversion from Ile-126 or Thr-126 of the wild-type virus to Ser-126 or Asn-126 in three carriers and conversion from Gly-145 to Arg-145 in three of them; clones with Asn-126 or Arg-145 were found in one carrier. All 12 clones from the other carrier had an insertion of 24 bp encoding an additional eight amino acids between Thr-123 and Cys-124. In addition, all or at least some of the HBV DNA clones from these carriers had in-phase deletions in the 5' terminus of the pre-S2 region. These results indicate that HBV escape mutants with mutations in the S gene affecting the expression of group-specific determinants would survive in some carriers after they seroconvert to antibody against surface antigen. Carriers with HBV escape mutants may transmit HBV either by donation of blood units without detectable surface antigen or through community-acquired infection, which would hardly be prevented by current hepatitis B immuneglobulin or vaccines.